Purification and characterization of a previously unreported form of cytochrome <i>P</i>-448 from the liver of 3-methylcholanthrene-pretreated rats

Seidel, Sharon L.; Shires, Thomas K.

doi:10.1042/bj2350859

Cited by 7 publications

(1 citation statement)

References 30 publications

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“…This is within the range (45,000 to 57,000) described for the majority of cytochromes P-450 (1,2,4,21,22,32). The spectral characteristics of the P-450so, are also similar to those of the other purified cytochromes P-450 (4) Cytochrome P-450S.Y is therefore the first substrate-free high-spin procaryotic P-450 to be reported, ranking among the limited number of high-spin cytochromes P-450, all isolated from either rat liver (24,31) or rabbit liver (5,13) sources.…”

Section: Discussionmentioning

confidence: 80%

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

1989

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A soybean flour-induced, soluble cytochrome P-450 (P-450,,.Y) was purified 130-fold to homogeneity from Streptomyces gyiseus. Native cytochrome P-450,,. is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450S exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450O had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin;NADP+ oxidoreductase, purified cytochrome P-450O, catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450SOYA.Streptomyces griseus (ATCC 13273) has the remarkable ability to catalyze the stereo-and regiospecific oxidation of a diverse array of xenobiotics. The types of reactions performed by S. griseus include aromatic, cyclic, and aliphatic hydroxylations, 0-, S-, and N-oxidations, C-C fission, epoxidation, and 0-and N-dealkylations. These transformations, which occur with compounds such as alkaloids (17,27), coumarins (25), rotenoids (28), chromenes (30), and other complex xenobiotics (7,26), parallel those performed by mammalian cytochromes P-450 (34, 36). However, the nature of the enzymatic system(s) employed by S. griseus for such diverse reactions has remained unresolved (26). The biotransformations catalyzed by S. griseus occur primarily following growth of the organism on a complex medium enriched with soybean flour (26). Recently, Sariaslani and Kunz (29) demonstrated that soybean flour and one of its isoflavonoid constituents, genistein, induce the synthesis of cytochrome P-450 in S. griseus and inferred a role for this enzyme in the biotransformation reactions performed by this organism. Here we report the purification and characterization of a soybean flour-induced cytochrome P-450 and demonstrate the transformation of two xenobiotic substrates, precocene II and 7-ethoxycoumarin, by purified preparations of this enzyme.( was determined by the method of Omura and Sato (23). Heme was analyzed as its reduced pyridine hemochrome derivative (10). High-pressure liquid chromatography (HPLC) was performed on a system consisting of an LKB 2152 controller, LKI3 2150 pump, and a Hewlett-Packard 1040A diode array detector.Cytochrome P-450.,y purification protocol. All procedures except steps 4 and 5 were carried out at 4°C. Glycerol (20% [vol/vol]) was included in all buffers following the salt precipitation step to prevent degradation of the P-450 to its inactive P-420 species. Protease inhibitors used in steps 1 to 3 to inhibit a wide range of protease activities were pepstatin (an acid protease inhibitor), leupeptin (a broad-range protease inhibitor), and phenylmethylsulfonyl fluoride (PMSF; a serine protease inhibitor). Fractions were stored between purification steps at -80°C with no discernable loss o...

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Section: Discussionmentioning

confidence: 80%

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

1989

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Purification and characterization of liver cytochrome P-446 isolated from protein energy malnourished rats

et al. 1988

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A liver cytochrome P-450 isozyme has been purified to homogeneity from protein-energy malnourished rats induced with beta-naphthoflavone (beta-NF). The purification steps included chromatography on DEAE-Sephadex-A-25, DEAE-cellulose (DE-53), hydroxylapatite (HA) and carboxymethyl-sephadex (CM) columns. The reduced carbon monoxide difference and absolute spectra showed a Soret peak at 446.5 nm. The wavelength maxima for the oxidized and reduced spectra were at 416 and 408 nm, respectively. Cytochrome P-446 appears to have a predominantly low spin ferric iron, migrates as a single band of molecular weight 56,000 in sodium dodecyl sulfate polyacrylamide gels and has a specific content of 14 nmol/mg of protein. P-446 oxidized various substrates at different rates in a reconstituted system with NADPH-cytochrome P-450 reductase and dilauroyl-phosphatidylcholine. In this system turnover rates for benzo[alpha]pyrene, testosterone and benzphetamine oxidation were: 81.10; 1.85 and 1.42 nmoles product/min/nmol P-446 respectively. While NH2 terminal amino acid sequence analysis of 18 of the first 20 residues suggests that the cytochrome P-446 isolated from malnourished rats is identical with form c, the catalytic activities suggest that this isozyme may be a more effective or efficient catalyst for some substrates.

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The cytochromes P-448—A unique family of enzymes involved in chemical toxicity and carcinogenesis

Ioannides

Parke

1987

Biochemical Pharmacology

120

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Purification and characterization of a previously unreported form of cytochrome P-448 from the liver of 3-methylcholanthrene-pretreated rats

Cited by 7 publications

References 30 publications

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

Purification and characterization of liver cytochrome P-446 isolated from protein energy malnourished rats

The cytochromes P-448—A unique family of enzymes involved in chemical toxicity and carcinogenesis

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